Cell division marker

ABSTRACT

This application relates to a newly identified animal cell structure, the midbody scar. This structure is a remnant of the midbody that is retained by one daughter cell following cytokinesis and persists through multiple subsequent cell cycles. The midbody scar can be useful as a marker of dividing cells or of a cell&#39;s replicative age.

CLAIM OF PRIORITY

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Patent Application Ser. No. 60/724,093, filed on Oct. 6,2005, the entire contents of which are hereby incorporated by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant numberGM51994 awarded by the National Institute of Health. The Government hascertain rights in the invention.

BACKGROUND

The mechanisms behind aging and longevity are a current topic ofinvestigation. Much progress has been made studying the genetics ofcellular aging in yeast. Yeast cell divisions produce a detectableremnant on the cell wall of the mother cell, known as a bud scar.Cellular aging in yeast is related to the number of daughter cellprogeny a cell has produced. By counting the number of bud scars on themother cell wall, the number of divisions, and thus the age of the cell,can be determined. A similar marker of previous cell divisions would beuseful for studies of cellular aging in mammals.

SUMMARY

The invention is based, inter alia, on the discovery of a cellularstructure in animal cells that is a marker of previous cell divisions.Following cell division, a remnant of the midbody ring is retained byone cell (typically the older or “parent” cell) and persists throughmultiple cell divisions, such that multiple midbody remnants canaccumulate in one cell. These remnants of the midbody ring, referred toherein as “midbody scars,” can be used to identify and isolate dividingcells, e.g., progenitor or stem cells, in a population. This applicationrelates to compositions and methods related to the newly discoveredmidbody scars.

In one aspect, the invention provides methods for detecting the presenceof a midbody scar in a mammalian cell. The methods include contacting amammalian cell suspected to have a midbody scar with an antibody orantigen-binding portion thereof that binds specifically to a polypeptidecomponent of a midbody scar selected from the group consisting of; anddetecting binding of the antibody to its antigen, wherein binding of theantibody indicates the presence of a midbody scar in the mammalian cell.

In some embodiments, the methods include contacting the mammalian cellwith two or more antibodies or antigen-binding portions thereof thateach bind specifically to a polypeptide component of a midbody scar, anddetecting binding of the two or more antibodies. Colocalized binding ofthe two or more of antibodies indicates the presence of a midbody scar.

In the methods described herein, the polypeptide component of themidbody scar can be, e.g., mitotic kinesin-like protein 1 (MKLP1);Epsilon tubulin (ε-tubulin); Centrosomal Protein 55 kDa (Cep55); andAurora B kinase.

In some embodiments, the cell is in interphase, e.g., in G1, S, or G2phase. Cells can be synchronized in interphase, e.g., in G1 (e.g.,arrested by serum starvation), S (by thymidine block), or in G2 (by flowout of S phase). In some embodiments, the mammalian cell is a fixedcell, and/or a cell in a pathology specimen. The cell can be isolatedcultured, or in tissue, e.g., in a fixed tissue sample. The cells ortissue can be from any organ in the human body, e.g., skin, heart,neural tissue, bone marrow, blood, liver, or lung, inter alia.

In another aspect, the invention provides methods for identifying adividing cell in a population of mammalian cells, e.g., a populationincluding both dividing cells and non-dividing cells. The methodsinclude obtaining a population of mammalian cells comprising at leastone cell suspected of being a dividing cell; and detecting a cell havingone or more midbody scars; a cell having one or more midbody scars is adividing cell. In some embodiments, the dividing cell is a cancerouscell. In some embodiments, the dividing cell is a stem or progenitorcell.

In some embodiments, the methods further include identifying cellshaving a desired number of midbody scars, e.g., 0 to 2 midbody scars percell, or 4 or more midbody scars per cell. Once cells having the desirednumber of midbody scars have been identified, they can be selected,e.g., for isolation, characterization, ablation, or other furthermanipulation. For example, cells selected and isolated by a methoddescribed herein can be expanded in culture under conditions sufficientto provide a population of dividing cells, e.g., a population comprisinga desired number of cells.

Identifying a cell with one or more midbody scars can be accomplished byoptically detecting the presence of a cellular structure havingmorphological characteristics of a midbody scar, e.g., using phasecontrast microscopy to analyze the cell. This analysis can be manual orautomated. Alternatively or in addition, cells with one or more midbodyscars can be identified by contacting the population of cells with adetectible antibody or antigen-binding portion thereof that bindsspecifically to a polypeptide component of a midbody scar; and detectingbinding of the detectible antibody to the polypeptide; in this case,binding of the antibody indicates the presence of a midbody scar.

In some embodiments, the methods described herein use an antibody orantigen-binding portion thereof is bound or linked to a detectablemoiety, and binding of the antibody to its antigen is detected bydetecting the detectable moiety. The detectible antibody or antigenbinding portion thereof can include, e.g., a fluorescent label, andidentifying a cell with one or more midbody scars is can be achieved bydetecting the presence of fluorescence from the detectible antibody orantigen binding portion thereof, e.g., using fluorescence microscopy,e.g., manually or using an automated system. In some embodiments, thedetectible antibody includes or is linked to an enzyme, which whenprovided with an appropriate substrate produces a product that isdetected.

In some embodiments, the population of cells is from a subject suspectedof having a proliferative disorder, e.g., a pathology specimen, and themethods can be used to diagnose a proliferative disease in a subject.For example, the methods can include determining the percentage of cellsin the sample that have a selected number of midbody scars; andcomparing the percentage of cells in the sample to a referencepercentage representing a sample from a subject that does not have aproliferative disorder; wherein the presence of a significantly highernumber of dividing cells as compared to the reference sample indicatesthat the subject has a proliferative disorder.

In further aspects, the invention features methods of determining thepresence of a midbody scar in an animal cell by contacting a cellsuspected to have a midbody scar with an agent that binds specificallyto a component of a midbody scar and detecting the agent, therebydetermining the presence of the midbody scar. In some embodiments, thecomponent of the midbody scar is MKLP1, ε-tubulin, Cep55, or Aurora Bkinase. In some embodiments, the agent comprises a detectable moiety,e.g., a fluorescent label, or a radioactive isotope. The label also canbe an enzyme, such as alkaline phosphatase or horseradish peroxidase,which when provided with an appropriate substrate produces a productthat is detectible.

In some embodiments, the agent is an antibody that binds specifically toa midbody scar or midbody scar component. In some embodiments, themethods include determining the presence of a midbody scar using phasemicroscopy. In various embodiments, the components are either withincells or exposed on the surface of the cells.

In other aspects, the invention features methods of selecting apopulation of cells with one or more, e.g., a desired number of, midbodyscars. In some embodiments, the methods include obtaining a populationof cells wherein midbody scars are optionally labeled with a detectablemoiety, detecting the midbody scars, and selecting cells with a desirednumber of midbody scars based on the presence or absence of the midbodyscars, e.g., based on the presence of absence of the detectable moiety.In some embodiments, the methods include obtaining a population of cellsand evaluating the cells using phase contrast microscopy, and selectingcells with a desired number of midbody scars based on the presence orabsence of a cell structure having the morphological features of amidbody scar. In some embodiments, the desired number of midbody scarsis 0, 1, 2, 3, 4, 5, 6, or 7 scars, e.g., between 0 and 2 or 4 or morescars. In some embodiments, the selection is done by an automatedsystem.

In further aspects, the invention features methods of determining orestimating the replicative age (i.e., the number of times the cell hasdivided to form two progeny) of an animal cell by detecting midbodyscars in an animal cell and counting the number of midbody scars percell, wherein the number of midbody scars per cell indicates the numberof divisions of the cell and thus the replicative age. In someembodiments, counting includes determining a level of a detectableentity per cell, e.g., a detectable entity bound covalently ornon-covalently to a midbody scar or midbody scar component, e.g., anantibody to a midbody ring protein as described herein.

In other aspects, the invention features methods of identifyingcompounds that bind to a midbody scar (or component thereof) byproviding a midbody scar, contacting the midbody scar with a testcompound, and determining whether the compound binds to the midbodyscar. In some embodiments, the midbody scar is isolated. In otherembodiments, the midbody scar is on or in a cell. The cell can be inculture or in a tissue or tumor, e.g., in a pathology sample, e.g.,including tissues known or suspected to include cells associated with aproliferative disorder. In some embodiments, determining whether thecompound binds to the midbody scar includes determining whether thecompound colocalizes with an agent described herein that binds to amidbody scar, e.g., an antibody that binds to a midbody ring protein asdescribed herein.

In further aspects, the invention features methods of identifyingcompounds that bind to midbody scar components by providing a midbodyscar component (e.g., MKLP1, ε-tubulin, Cep55, or Aurora B kinase, orfragments thereof), contacting the midbody scar component with a testcompound, and determining whether the test compound binds to the midbodyscar component. In some embodiments, the midbody scar component isisolated, e.g., an isolated protein. In other embodiments, the midbodyscar component is in a midbody scar, e.g., is part of an isolatedmidbody scar. In other embodiments, the midbody scar component is in acell, e.g., is part of a midbody scar in a cell or in a tissue.

In other aspects, the invention features methods and compounds fortargeting therapeutic or diagnostic compositions to cells that containmidbody scars. The methods include conjugating a therapeutic ordiagnostic moiety to a compound that binds to a midbody scar on a cellto produce a therapeutic or diagnostic composition, and administeringthe therapeutic or diagnostic composition to a subject.

In some aspects, the invention features isolated midbody scars frommammalian cells and tissues, e.g., normal tissues, pathology tissues(e.g., known or suspected to include cancerous cells), and blood, e.g.,from a mammal such as a human. The isolated midbody scars typicallyinclude certain proteins such as mitotic kinesin-like protein 1 (MKLP1),Epsilon Tubulin (ε-tubulin), Cep55, and/or Aurora B kinase. In someembodiments, the midbody scars do not include γ-tubulin or centriolin.In some embodiments, the isolated midbody scars are from interphasecells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

This patent and the corresponding application file contain at least onedrawing executed in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIGS. 1A-1E are a series of micrographs depicting a mitotic kinesin-likeprotein 1 (MKLP1)-positive ring localized at the midbody duringcytokinesis. FIG 1A is a phase contrast picture. FIG. 1B depicts MKLP1staining. FIG. 1C depicts γ-tubulin staining. FIG 1D depicts DAPIstaining of DNA. FIG. 1E is a merged combination of FIGS. 1B-1D, inwhich MKLP1 staining is pseudo-colored in red in the original, γ-tubulinstaining is pseudo-colored in green in the original, and DAPI stainingis pseudo-colored in blue in the original. The midbody structure isenlarged in the insets.

FIGS. 2A-2E are a series of micrographs depicting a persistent midbodyscar in a prophase cell. FIG. 2A is a phase contrast picture. FIG. 2Bdepicts MKLP1 staining. FIG. 2C depicts γ-tubulin staining. FIG. 2Ddepicts DAPI staining of DNA. FIG. 2E is a merged combination of FIGS.2B-2D, in which MKLP1 staining is pseudo-colored in red in the original,γ-tubulin staining is pseudo-colored in green in the original, and DAPIstaining is pseudo-colored in blue in the original. The midbody scar isenlarged in the insets.

FIGS. 3A ₁-3C₅ are a series of micrographs depicting a persistentmidbody scar during mitosis. FIGS. 3A ₁-3A₅ depict metaphase. FIGS. 3B₁-3B₅ depict anaphase. FIGS. 3C ₁-3C₅ depict telophase. FIGS. 3A ₁-3B₁,and 3C₁ show phase contrast. FIGS. 3A ₂, 3B₂, and 3C₂ depict γ-tubulinstaining. FIGS. 3A ₃, 3B₃, and 3C₃ depict MKLP1 staining. FIGS. 3A ₄,3B₄, and 3C₄ depict DAPI staining of DNA. FIGS. 3A ₅, 3B₅, and 3C₅depict a merged version of the three previous figures, with γ-tubulinpseudo-colored in green in the original, MKLP1 pseudo-colored in red inthe original, and DAPI pseudo-colored in blue in the original. Midbodyscar(s) can be observed at metaphase (3A₃; 3A₅, red in the original),anaphase (3B₃; 3B₅, red in the original), and telophase (3C₃; 3C₅, redin the original). The midbody scars are enlarged in the insets.

FIGS. 4A-4D are a series of fluorescence micrographs depicting a cellwith multiple MKLP1-positive midbody scars. FIG. 4A depicts staining ofα-tubulin. FIG. 4B depicts staining of MKLP1. FIG. 4C depicts DAPIstaining of DNA. FIG. 4D is the merge of FIGS. 4A-4C, with α-tubulinpseudo-colored in green in the original, MKLP1 pseudo-colored in red inthe original, and DAPI pseudo-colored in blue in the original. Fourmidbody scars are visible in one cell, and an adjacent cell contains onemidbody scar.

FIG. 5 is a photomicrograph of H9 human embryonic stem cells, withstaining for nuclei and MKLP-1.

DETAILED DESCRIPTION

This invention includes compositions and methods related to a newlydiscovered cellular structure, the midbody scar. This structure ispreferentially segregated to one cell during cytokinesis and persiststhrough multiple cell divisions, such that a cell can accumulatemultiple midbody scars, potentially accumulating one for each time itdivides, e.g., as it “ages.” These scars can also be used as markers toidentify stem cells in a population (see below) and can be used toidentify, isolate, and characterize various proliferating cells in humantissues and cell samples for use in numerous diagnostic and therapeuticmethods.

Cytokinesis is a fundamental process that results in the division of asingle cell with replicated chromosomes into two daughter cells withidentical genetic composition (see, e.g., Glotzer, 2001, Annu. Rev. CellDev. Biol., 17:351-386; Glotzer, 2005, Science, 307:1735-1739; andGuertin, 2002, Microbiol. Mol. Biol. Rev., 66:155-178). Duringcytokinesis, a phase-dense material known as the midbody forms betweenthe two daughter cells. This midbody has been shown to be involved inrecruitment of secretory vesicles during daughter cell abscission(Gromley et al., 2005, Cell, 123:1-13).

Midbody Scars

This application describes the discovery of a new cellular structurerelated to the midbody. This structure, the midbody scar, can persistthrough several subsequent cell divisions, such that multiple midbodyscars can be detected in some cells (Examples 1-4). As many as sixmidbody scars have been detected in a single cell, although the upperlimit on number of midbody scars in a cell can be several times higher.

The midbody scars form when, following cytokinesis, the midbody ispreferentially segregated to one of the resulting daughter cells. Theother daughter cell receives no detectable trace of the midbody. Asdemonstrated herein, the midbody scar can segregate preferentially,possibly based on the existence of a previous scar in the cell. When ayoung cell with no midbody scar divides, the midbody scar may besegregated randomly. Importantly, when a stem or progenitor celldivides, e.g., a pluripotent or immortal cell, the midbody scar willgenerally segregate with the original stem cell, and not with the newlyformed daughter cell, e.g., the cell that differentiates. Thus, themidbody scars are markers of stem and progenitor cells, and can be usedto identify, target, and isolate these cells.

The midbody scar structure is localized adjacent to the plasma membrane,and is made up of several proteins, including those listed in Table 1.The midbody scar contains many of the proteins present in the midbody,with some exceptions; for example, in some embodiments, γ-tubulin, whichis a component of the midbody, is not present in the midbody scar. Thepolypeptide components of midbody scars include kinases, otherregulatory proteins, and structural proteins. The accumulation ofmidbody scar structures, including such regulatory proteins, may affectcell division processes. TABLE 1 Exemplary Polypeptide Components ofMammalian Midbody Scars EntrezGene Protein name database ID Also knownas mitotic kinesin-like GeneID: 9493 KIF-23; CHO1; KNSL5; and protein 1(MKLP1) MKLP-1 Epsilon tubulin GeneID: 51175 TUBE1, TUBE; FLJ22589; and(ε-tubulin) dJ142L7.2 Centrosomal Protein GeneID: 55165 URCC6; C10orf3;and FLJ10540 55 kDa (Cep55) Aurora B kinase GeneID: 9212 AIK2; AIM1;ARK2; AurB; IPL1; STK5; AIM-1; and STK12Isolation of Midbody Scars

Midbody scars are stable structures that can persist in cells throughmultiple cell cycles. Accordingly, the midbody scar structure can beisolated, in whole or in part, by common cell biology techniques.Methods of purification of proteins and protein complexes are known toone of skill in the art. The midbody scar can be purified underconditions suitable to keep the complex intact. For example, themembranes can be solubilized with a non-ionic detergent, such as Triton™X-100 and the scars isolated on a density gradient and/or antibodyaffinity matrix. General methods of protein purification are described,e.g., in Scopes, Protein Purification: Principles and Practice, Springer(3d ed. 1993).

Methods of midbody purification are known (see, e.g., Kuriyama andEnsrud, 1999, Methods Cell Biol., 61:233-244; and Skop et al., 2004,Science, 305:61-66). These methods can be adapted for isolation ofmidbody scars. For example, Skop et al. (supra) describes thepurification of midbodies from a synchronized population of mitoticcells. For example, the methods of Skop et al. (supra) can be used withinterphase cells to obtain a preparation enriched in midbody scars. Insome embodiments, a synchronized population of non-mitotic cells can beused to isolate midbody scars. In some embodiments, midbodies can beseparated from midbody scars with a compound that binds specifically toa component of midbodies that is not found in midbody scars, e.g.,γ-tubulin. Midbody scars can also be isolated or enriched further by thesame method (e.g., using antibody-bound affinity purification beads ormatrices, e.g., containing antibodies to a polypeptide component of themidbody scar as described herein).

Isolated midbody scars can be used to determine the identity of midbodyscar or associated proteins. The protein complex can be dissociated bytreatment with an ionic detergent, such as SDS, and the individualproteins can be isolated and identified, e.g., by mass spectrometry.Regulatory proteins, e.g., kinases, that are part of the midbody scarmay be involved in cellular aging processes or in cell immortality (stemcells). Because the midbody scar is a remnant of the midbody, it islikely to contain many of the proteins present in the midbody (see,e.g., Skop et al., 2004, Science, 305:61-66). Additionally, there may beproteins present in the midbody scar that are not found in the midbody.

Detection of Midbody Scars

The midbody scars can be detected by a variety of methods. Midbody scarscan be detected to determine a cell's replicative age, to select cellpopulations based on the presence of one or more midbody scars, toidentify and/or target cells based on the presence of one or moremidbody scars, and to study the characteristics of cells with midbodyscars.

Midbody scars can be detected by using a molecule that binds to themidbody scar, e.g., an antibody that specifically binds to a midbodyscar polypeptide or an antigenic fragment thereof, e.g., MKLP1,ε-tubulin, Cep55, or Aurora B kinase. A number of antibodies suitablefor use in the methods described herein are known in the art and/or arecommercially available. For example, anti-MKLP1 and anti-Aurora Bantibodies are available from BD Biosciences (San Jose, Calif.) andSanta Cruz Biotechnology Inc. (Santa Cruz, Calif.). Antibodies toε-tubulin are described in Chang and Stearns, Nature Cell Biology2:30-35 (2000), and are available from Sigma-Aldrich (St. Louis, Mo.).Antibodies to Cep55 are described in Fabbro et al., Dev. Cell 9:477-488(2005), and Zhao et al. Mol. Biol. Cell. 17:3881-3896 (2006), and areavailable from Abnova Corp. (Taipei City, Taiwan). The antibody can be,for example, an antigen-binding fragment of a full-length antibody. Theantibody can also be a CDR-grafted antibody, diabody, triabody,minibody, humanized antibody, deimmunized antibody, or an antigenbinding fragments thereof.

In some embodiments, the antibody is conjugated to a detectable entity,e.g., a fluorescent moiety, a radioactive isotope, an enzyme, a contrastagent, or a magnetic particle. In some embodiments, the antibody is notlabeled with a detectable entity, but can itself be bound by a compoundcontaining a detectable entity, e.g., an antibody to the compound or amember of a binding pair (e.g., avidin and biotin). When live cells areused, the midbody scar binding moiety or agent may be chosen to bind toan extracellular exposed portion of the midbody scar, e.g., a componentof the scar that has some portion that is exposed on the surface of thecell. Alternately, the midbody scar binding moiety or agent can bedesigned to cross the cell membrane to bind to a portion of the midbodyscar not exposed extracellularly (i.e., an intracellular portion). Forexample, an antibody (or other binding agent) that binds to anintracellular portion of the midbody scar can include a cellinternalization peptide, such as Antennapedia, HIV-derived TAT peptide,penetratins, transportans, or hCT derived cell-penetrating peptides,see, e.g., Caron et al., (2001) Mol. Ther. 3(3):310-8; Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla. 2002); El-Andaloussi et al., (2005) Curr Pharm Des.11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci.62(16):1839-49, or can be packaged inside a carrier that allows theantibody to cross the membrane, e.g., a vesicle or liposome.

In other embodiments, a polypeptide that is a component of the midbodyscar can be produced as a fusion protein that includes a fluorescentpolypeptide, such as GFP. Such fluorescent fusion proteins can be chosensuch that the midbody scar polypeptide still functions in the midbodyscar complex. Such fusion proteins can be introduced into cells and usedto mark midbody scars, study them in living cells, and isolate thembased on the presence of the fluorescence marker.

Detection of midbody scars in a cell allows the determination of thereplicative age of the cell. As a cell divides, it can accumulate amidbody scar with each cell cycle, therefore, cells with higher numbersof midbody scars are those that have undergone a higher number of celldivisions compared with cells having fewer or lacking scars.

In some embodiments, the relative number of midbody scars can beapproximated by the relative level of signal, e.g., a fluorescent signal(e.g., from MKLP1-GFP or a fluorescently labeled antibody to a midbodyscar protein), in the cells. In this system, interphase cells with twomidbody scars will have approximately twice the signal as interphasecells with one midbody scar. Based on the relative level of signal, thecells can be sorted using routine methods to select a population that isenriched for the presence of midbody scars, e.g., for a specific numberor range of numbers of midbody scars. Isolation of a homogeneouspopulation of cells (e.g., all dividing cells, e.g., all stem orprogenitor cells) can be useful, e.g., for achieving a standardizedresponse to drugs or other cell stimuli, or for preparing a populationof stem cells for use in other applications, e.g., therapeutic ordiagnostic applications as described herein.

Stem Cells

Stem cells are primal cells that, upon division, can give rise both toanother stem cell and a cell that will differentiate and ultimately die(see, e.g., Zipori, Nat. Rev. Genet. 5:873-878 (2004)). Totipotent stemcells are produced from the fusion of an egg and sperm cell. Cellsproduced by the first few divisions of the fertilized egg cell are alsototipotent. These cells can differentiate into embryonic andextraembryonic cell types. Pluripotent stem cells are the descendants oftotipotent cells and can differentiate into cells derived from the threegerm layers. Multipotent stem cells can produce only cells of a closelyrelated family of cells (e.g. hematopoietic stem cells differentiateinto red blood cells, white blood cells, or platelets). Unipotent cellscan produce only one cell type, but have the property of self-renewal,which distinguishes them from non-stem cells. Stem cells are nowbelieved to be present, though extremely scarce, in most tissues of themammalian body, including the adult body.

Presently, there are no good universal markers for stem cells. CD44 is awidely used marker, but does not detect all stem cell populations. It isreasonable to believe that midbody scars are present in all stem cellpopulations and can therefore be used as a universal stem cell marker.Therefore, midbody scars can be used to identify stem cellsprospectively, thereby potentially avoiding the otherwise necessarytasks of growing cells, cloning cells, and passing cells through mice toidentify stem cell populations. The use of midbody scars as a markerallows the identification of stem cells in various organs andenvironments.

An emerging cancer stem cell theory posits that cancer develops fromstem cells rather than from de-differentiated cells as previouslybelieved (see, e.g., Marx, Science 301:1308-1310 (2003); Bell and VanZant, Oncogene 23:7290-7296 (2004); Jordan et al., N. Eng. J. Med.355:1253 (2006)). This new theory has gained considerable acceptance.Such stem cells should have midbody scars that would serve as markersfor tumor stem cells. These structures could also serve as targets fortumor cell killing. Moreover, isolated midbody scars could becharacterized to identify novel markers for therapeutic targets.

Identification and Selection of Dividing Cells

Dividing cells, such as stem cells and cancer cells, can be identifiedby the presence and/or number of midbody scars accumulated during orafter each division cycle. Thus, midbody scars can be detected to selecta population of cells that are dividing. For example, cells can beselected that have relatively few (e.g., 0 to 2) midbody scars or thathave relatively many (e.g., 4 or more) midbody scars. This selection canbe performed manually, or can be automated, e.g., by flow cytometry cellsorting, e.g., fluorescence activated cell sorting (FACS) for cells withmidbody scars that are labeled with fluorescent moieties or magneticactivated cell sorting (MACS) for cells with midbody scars that arelabeled with magnetic beads; or optical image-based cell sorting, basedon the detection of cellular structures with the morphological featuresof midbody rings, e.g., using phase contrast microscopy. At highmagnification (i.e., over about 60×-100×), small bright rings of about 2μm are visible.

Once the cells have been identified, the cells can be selected eitherwith a pick-to-remove (i.e., select the cells that lack midbody scarsfor removal, and remove them, e.g., by aspiration with culture media orby laser ablation using standard methods) or a pick-to-keep (i.e.,select the cells that have a selected number of midbody scars and movethem to a new culture vessel) scheme. Dividing cells such as stem orprogenitor cells can be maintained in culture using methods known in theart, e.g., including the use of specialized stem cell culture mediaincluding the BMP antagonist noggin and basic fibroblast growth factor(bFGF) and/or feeder cells such as primary or immortalized mouseembryonic fibroblasts (MEFs). See, for example, Xu et al., Nat. Meth.2(3):185-190 (2005), U.S. Pat. No. 7,005,252, and the protocols of theWiCell Research Institute, Inc., available on the world wide web atWiCell.org. When passaging the cells or otherwise treating the cells torelease them from a culture substrate, it is desirable to use a methodthat is not destructive of extracellular proteins; thus, the use ofAccutase™ (a gently enzyme cell detachment medium, from Innovative CellTechnologies, Inc.), or removal/reduction in levels of divalent cationsusing chelators such as EGTA or EDTA, is preferred over the use ofstrong proteases such as trypsin, which can disrupt the midbody scarsboth outside and inside cells.

As one example, a population of cells can be diluted and plated into amultiwell plate or other suitable vessel in such a manner that only oneor a small number of cells is plated in each well. The wells can beevaluated, e.g., manually or by automated means, for the presence ofcells with one or more, e.g., a selected number of, midbody scars. Thosewells having cells with midbody scars can be selected for clonalexpansion, and cultured using methods known in the art. For example, aclonal population of stem cells can be produced using a method describedherein and culture methods known in the art for the maintenance andpropagation of stem cells.

Screening Methods

The invention provides methods for identifying compounds, e.g., smallorganic or inorganic molecules (e.g., those with a molecular weight ofless than 1,000 Da), oligopeptides, oligonucleotides, or carbohydrates,capable of binding to midbody scars or midbody scar polypeptides. Suchcompounds can be useful in methods relating to studying and/or alteringaging and longevity.

In certain embodiments, screening methods of the present inventionutilize libraries of test compounds. As used herein, a “test compound”can be any chemical compound, for example, a macromolecule (e.g., apolypeptide, a protein complex, glycoprotein, or a nucleic acid) or asmall molecule (e.g., an amino acid, a nucleotide, an organic orinorganic compound). A test compound can have a formula weight of lessthan about 100,000 grams per mole, less than about 50,000 grams permole, less than about 10,000 grams per mole, less than 5,000 grams permole, less than 1,000 grams per mole, or less than about 500 grams permole. The test compound can be naturally occurring (e.g., an herb or anatural product), synthetic, or can include both natural and syntheticcomponents. Examples of test compounds include proteins, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, and organic or inorganic compounds, e.g., heteroorganic ororganometallic compounds.

Test compounds can be screened individually or in parallel. An exampleof parallel screening is a high throughput drug screen of largelibraries of chemicals. Such libraries of candidate compounds can begenerated or purchased, e.g., from Chembridge Corp. (San Diego, Calif.).Libraries can be designed to cover a diverse range of compounds. Forexample, a library can include 500, 1000, 10,000, 50,000, 100,000, ormore unique compounds. Alternatively, prior experimentation andanecdotal evidence can suggest a class or category of compounds ofenhanced potential. A library can be designed and synthesized to coversuch a class of chemicals.

The synthesis of combinatorial libraries is well known in the art andhas been reviewed (see, e.g., Gordon et al., J. Med. Chem. (1994)37:1385-1401; DeWitt and Czamik, Acc. Chem. Res. (1996) 29:114;Armstrong et al., Acc. Chem. Res. (1996) 29:123; Ellman, Acc. Chem. Res.(1996) 29:132; Gordon et al., Acc. Chem. Res. (1996) 29:144; Lowe, Chem.Soc. Rev. (1995) 309; Blondelle et al., Trends Anal. Chem. (1995) 14:83;Chen et al., J. Am. Chem. Soc. (1994) 116:2661; U.S. Pat. Nos.5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. WO92/10092,WO93/09668, WO91/07087, WO93/20242, WO94/08051).

Libraries of compounds can be prepared according to a variety of methodsknown in the art. For example, a “split-pool” strategy can beimplemented in the following way: beads of a functionalized polymericsupport are placed in a plurality of reaction vessels; a variety ofpolymeric supports suitable for solid-phase peptide synthesis are known,and some are commercially available (for examples, see, e.g., Bodansky“Principles of Peptide Synthesis”, 2nd edition, Springer-Verlag, Berlin(1993)). To each aliquot of beads is added a solution of a differentactivated amino acid, and the reactions are allowed to proceed to yielda plurality of immobilized amino acids, one in each reaction vessel. Thealiquots of derivatized beads are then washed, “pooled” (i.e.,recombined), and the pool of beads is again divided, with each aliquotbeing placed in a separate reaction vessel. Another activated amino acidis then added to each aliquot of beads. The cycle of synthesis isrepeated until a desired peptide length is obtained.

The amino acid residues added at each synthesis cycle can be randomlyselected; alternatively, amino acids can be selected to provide a“biased” library, e.g., a library in which certain portions of theinhibitor are selected non-randomly, e.g., to provide an inhibitorhaving known structural similarity or homology to a known peptidecapable of interacting with an antibody, e.g., the an anti-idiotypicantibody antigen binding site. It will be appreciated that a widevariety of peptidic, peptidomimetic, or non-peptidic compounds can bereadily generated in this way.

The “split-pool” strategy can result in a library of peptides, e.g.,modulators, which can be used to prepare a library of test compounds ofthe invention. In another illustrative synthesis, a “diversomer library”is created by the method of DeWitt et al., (Proc. Natl. Acad. Sci. USA,90:6909-13 (1993)). Other synthesis methods, including the “tea-bag”technique of Houghten (see, e.g., Houghten et al., Nature, 354:84-86(1991)) can also be used to synthesize libraries of compounds accordingto the subject invention.

Libraries of compounds can be screened to determine whether any membersof the library have a desired activity, and, if so, to identify theactive species. Methods of screening combinatorial libraries have beendescribed (see, e.g., Gordon et al., J. Med. Chem., supra). Solublecompound libraries can be screened by affinity chromatography with anappropriate receptor to isolate ligands for the receptor, followed byidentification of the isolated ligands by conventional techniques (e.g.,mass spectrometry, NMR, and the like). Immobilized compounds can bescreened by contacting the compounds with a soluble receptor;preferably, the soluble receptor is conjugated to a label (e.g.,fluorophores, colorimetric enzymes, radioisotopes, luminescentcompounds, and the like) that can be detected to indicate ligandbinding. Alternatively, immobilized compounds can be selectivelyreleased and allowed to diffuse through a membrane to interact with areceptor. Exemplary assays useful for screening libraries of testcompounds are described herein.

The invention provides methods for identifying compounds capable ofbinding to midbody scars or midbody scar polypeptides (e.g., MKLP1 orAurora B kinase).

In some embodiments, the assay is a cell-based assay in which a cellthat includes at least one midbody scar is contacted with a testcompound, and the ability of the test compound to bind to the midbodyscar is determined, e.g., by binding and localization to the midbodyscar. The cell, for example, can be of mammalian origin, e.g., murine,rat, or human origin.

The ability of a compound to interact with a midbody scar or midbodyscar polypeptide with or without the labeling of any of the interactantscan be evaluated. For example, a microphysiometer can be used to detectthe interaction of a compound with a midbody scar or midbody scarpolypeptide without labeling either the compound or the midbody scar ormidbody scar polypeptide (McConnell et al., 1992, Science,257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor®)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and a midbody scar ormidbody scar polypeptide.

Soluble and/or membrane-bound forms of midbody scars and midbody scarpolypeptides can be used in cell-free assays as described herein. Whenmembrane-bound forms of the protein are used, it may be desirable toutilize a solubilizing agent. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of isolatedmidbody scars and one or more test compounds under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence resonance energy transfer (FRET) (see, for example,Lakowicz et al., U.S. Pat. No. 5,631,169 and Stavrianopoulos et al.,U.S. Pat. No. 4,868,103). A fluorophore label on the first ‘donor’molecule is selected such that its emitted fluorescent energy will beabsorbed by a fluorescent label on a second ‘acceptor’ molecule, whichin turn is able to fluoresce due to the absorbed energy. Alternately,the ‘donor’ protein molecule may simply utilize the natural fluorescentenergy of tryptophan residues. Labels are chosen that emit differentwavelengths of light, such that the ‘acceptor’ molecule label may bedifferentiated from that of the ‘donor.’ Since the efficiency of energytransfer between the labels is related to the distance separating themolecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. A FRET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

In another embodiment, determining the ability of a midbody scar ormidbody scar polypeptide to bind to a target molecule can beaccomplished using real-time Biomolecular Interaction Analysis (BIA)(e.g., Sjolander et al., 1991, Anal. Chem., 63:2338-2345 and Szabo etal., 1995, Curr. Opin. Struct. Biol., 5:699-705). “Surface plasmonresonance” or “BIA” detects biospecific interactions in real time,without labeling any of the interactants (e.g., BIAcore). Changes in themass at the binding surface (indicative of a binding event) result inalterations of the refractive index of light near the surface (theoptical phenomenon of surface plasmon resonance (SPR)), resulting in adetectable signal that can be used as an indication of real-timereactions between biological molecules.

In one embodiment, the target midbody scar, or midbody scar polypeptide,or the test substance is anchored onto a solid phase. The target midbodyscar or midbody scar polypeptide/test compound complexes anchored on thesolid phase can be detected at the end of the reaction. The targetmidbody scar or midbody scar polypeptide can be anchored onto a solidsurface, and the test compound, which is not anchored, can be labeled,either directly or indirectly, with detectable labels discussed herein.

Binding compounds are screened by identifying from a group of testcompounds those that bind to a midbody scar or midbody scar polypeptide.Binding of a test compound to a midbody scar or midbody scar polypeptidecan be detected, for example, in vitro by reversibly or irreversiblyimmobilizing the test compound(s) on a substrate, e.g., the surface of awell of a 96-well polystyrene microtiter plate. Methods for immobilizingpolypeptides and other small molecules are well known in the art. Forexample, microtiter plates can be coated with midbody scars or a midbodyscar polypeptide by adding the midbody scar polypeptide in a solution(typically, at a concentration of 0.05 to 1 mg/ml in a volume of 1-100μl water or buffer) to each well, and incubating the plates at roomtemperature to 37° C. for a given amount of time, e.g., for 0.1 to 36hours. Polypeptides not bound to the plate can be removed, e.g., bypouring excess solution from the plate, and then washing the plate (onceor repeatedly) with water or a buffer. The plate can then be washed witha buffer that lacks the bound polypeptide.

To block the free protein-binding sites on the plates, plates can beblocked with a protein that is unrelated to the bound polypeptide. Forexample, 300 μl of bovine serum albumin (BSA) at a concentration of 2mg/ml in Tris-HCl can be used. Suitable substrates include thosesubstrates that contain a defined cross-linking chemistry (e.g., plasticsubstrates, such as polystyrene, styrene, or polypropylene substratesfrom Corning Costar Corp. (Cambridge, Mass.), for example). If desired,a beaded particle, e.g., beaded agarose or beaded Sepharose®, can beused as the substrate. Test compounds can then be added to the coatedplate and allowed to bind to the midbody scar or midbody scarpolypeptide (e.g., at 37° C. for 0.5-12 hours). The plate can then bewashed as described above.

Binding of a midbody scar or midbody scar polypeptide to a secondcompound, e.g., a test compound described above, can be detected by anyof a variety of art-known methods. For example, an antibody thatspecifically binds to a midbody scar polypeptide (e.g., an anti-MKLP1 orAurora B kinase antibody) can be used in an immunoassay. If desired, theantibody can be labeled (e.g., fluorescently or with a radioisotope) anddetected directly (see, e.g., West and McMahon, J. Cell Biol., 74:264,(1977)). Alternatively, a second antibody can be used for detection(e.g., a labeled antibody that binds to the Fc portion of theanti-midbody scar polypeptide antibody).

In an alternative detection method, the midbody scar or midbody scarpolypeptide is labeled (e.g., with a radioisotope, fluorophore,chromophore, or the like), and the label is detected. In still anothermethod, a midbody scar polypeptide is produced as a fusion protein witha protein that can be detected optically, e.g., green fluorescentprotein (which can be detected using an appropriate light source). In analternative method, the polypeptide is produced as a fusion protein withan enzyme having a detectable enzymatic activity, such as horseradishperoxidase, alkaline phosphatase, β-galactosidase, or glucose oxidase.Genes encoding all of these enzymes have been cloned and are availablefor use by skilled practitioners. If desired, the fusion protein caninclude an antigen, which can be detected and measured with a polyclonalor monoclonal antibody using conventional methods. Suitable antigensinclude enzymes (e.g., horse radish peroxidase, alkaline phosphatase,and βgalactosidase) and non-enzymatic polypeptides (e.g., serumproteins, such as BSA and globulins, and milk proteins, such ascaseins).

In various methods for identifying polypeptides, e.g., testpolypeptides, that bind to a midbody scar polypeptide, the conventionaltwo-hybrid assays of protein/protein interactions can be used (see e.g.,Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578 (1991); Fields et al.,U.S. Pat. No. 5,283,173; Fields and Song, Nature, 340:245 (1989); LeDouarin et al., Nucleic Acids Research, 23:876 (1995); Vidal et al.,Proc. Natl. Acad. Sci. USA, 93:10315-10320 (1996); and White, Proc.Natl. Acad. Sci. USA, 93:10001-10003 (1996)). Typically, two-hybridmethods involve reconstitution of two separable domains of atranscription factor. One fusion protein contains the midbody scarpolypeptide fused to either a transactivator domain or DNA bindingdomain of a transcription factor (e.g., of Gal4). The other fusionprotein contains a test polypeptide fused to either the DNA bindingdomain or a transactivator domain of a transcription factor. Oncebrought together in a single cell (e.g., a yeast cell or mammaliancell), one of the fusion proteins contains the transactivator domain andthe other fusion protein contains the DNA binding domain. Therefore,binding of the midbody scar polypeptide to the test polypeptidereconstitutes the transcription factor. Reconstitution of thetranscription factor can be detected by detecting expression of a gene(i.e., a reporter gene) that is operably linked to a DNA sequence thatis bound by the DNA binding domain of the transcription factor. Kits forpracticing various two-hybrid methods are commercially available (e.g.,from Clontech; Palo Alto, Calif.).

Additionally, polypeptides involved in the removal or degradation ofmidbody scars can be identified.

Antibodies

Full length proteins or polypeptides of the midbody scar, or immunogenicfragments or analogs thereof, can be used to raise antibodies useful inthe methods described herein; such polypeptides can be produced byrecombinant techniques or synthesized (see, for example, Stewart, “SolidPhase Peptide Synthesis,” Freeman (1968); Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory, Cold SpringHarbor, N.Y. (1989); Ausubel et al. (Eds.), Current Protocolsin—Molecular Biology, John Wiley & Sons, New York, N.Y., 1999 andpreceding editions; and U.S. Pat. No. 4,237,224). In general, thepeptides can be coupled to a carrier protein, such as KLH, as describedin Ausubel et al., supra, mixed with an adjuvant, and injected into ahost mammal. Antibodies can be purified by peptide antigen affinitychromatography.

In particular, various host animals can be immunized by injection with apolypeptide of the invention. Host animals include rabbits, mice, guineapigs, and rats. Various adjuvants that can be used to increase theimmunological response depend on the host species and include Freund'sadjuvant (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Useful human adjuvants include BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies areheterogeneous populations of antibody molecules that are contained inthe sera of the immunized animals.

Antibodies for use in the new methods include polyclonal antibodies and,in addition, monoclonal antibodies, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′)₂ fragments, CDR-graftedantibodies, deimmunized antibodies, molecules produced using a Fabexpression library, and antigen binding fragments thereof.

Monoclonal antibodies (mAbs), which are homogeneous populations ofantibodies to a particular antigen, can be prepared using thepolypeptides of the invention described above and standard hybridomatechnology (see, for example, Kohler et al., Nature, 256:495, 1975;Kohler et al., Eur. J. Immunol., 6:511, 1976; Kohler et al., Eur. J.Immunol., 6:292, 1976; Hammerling et al., “Monoclonal Antibodies and TCell Hybridomas,” Elsevier, N.Y., 1981; Ausubel et al., supra).

In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described in Kohler et al. (Nature,256:495, 1975, and U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kosbor et al., Immunology Today, 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA, 80:2026, 1983); and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. Hybridomas producing mAbs may be cultivated in vitro or invivo. The ability to produce high titers of mAbs in vivo makes this aparticularly useful method of production.

Once produced, polyclonal or monoclonal antibodies are tested forspecific recognition of midbody scar polypeptides, or fragment thereof,by Western blot or immunoprecipitation analysis by standard methods(e.g., as described in Ausubel et al., supra). Antibodies thatspecifically recognize and bind to midbody scar proteins, or fragmentsthereof, are useful in the methods described herein. For example, suchantibodies can be used in an immunoassay to monitor a midbody scarprotein, or fragment thereof, in mammalian cells (for example, todetermine the amount or subcellular location of a midbody scar protein,or fragment thereof).

In some embodiments, antibodies are produced using fragments of themidbody scar polypeptides that lie outside highly conserved regions andappear likely to be antigenic, by criteria such as high frequency ofcharged residues. In one specific example, such fragments are generatedby standard techniques of PCR, and are then cloned into the pGEXexpression vector (Ausubel et al., supra). Fusion proteins are expressedin E. coli and purified using a glutathione agarose affinity matrix asdescribed in Ausubel, et al., supra.

In some cases it may be desirable to minimize the potential problems oflow affinity or specificity of antisera. In such circumstances, two orthree fusions can be generated for each protein, and each fusion can beinjected into at least two rabbits. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.

In other embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al.,Nature, 314:452, 1984) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration are oftenpossible than with non-human antibodies. Modifications such aslipidation can be used to stabilize antibodies and to enhance uptake andtissue penetration (e.g., into the brain). A method for lipidation ofantibodies is described by Cruikshank et al. (J. Acquir. Immune Defic.Syndr., 14:193, 1997).

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against midbody scarproteins, or fragments thereof. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to midbody scar polypeptides can, in turn, be used togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art (see, e.g., Greenspan et al., FASEB J., 7:437, 1993;Nissinoff, J. Immunol., 147:2429, 1991). For example, antibodies thatbind to a midbody scar polypeptide and competitively inhibit the bindingof a binding partner of that midbody scar polypeptide can be used togenerate anti-idiotypes that resemble a binding partner binding domainof the protein and, therefore, bind and neutralize a binding partner ofthe protein. Such neutralizing anti-idiotypic antibodies or Fabfragments of such anti-idiotypic antibodies can be used in therapeuticregimens.

Antibodies can be humanized by methods known in the art. For example,monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics, 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and5,569,825).

Therapeutic and Diagnostic Uses

Antibodies to midbody scar proteins can be used in therapeutic ordiagnostic methods as described herein, e.g., to target therapeutics ordiagnostic moieties to a subpopulation of cells with midbody scars.Identification of cells having midbody scars can also enable thetargeting of other therapeutic modalities, e.g., laser ablation based onthe presence of midbody scars to remove hyperproliferating stem cellsfrom tissues such as tumors, or skin lesions such as psoriatic lesions,and thereby treat proliferative disease.

For example, the detection of midbody scars can be used to detectproliferating cells in a sample suspected of containing canceroustissues. In some embodiments, the cells in the sample are fixed andcontacted with one or more antibodies to a polypeptide component of amidbody scar. One of skill in the art would be able to determine whetherthe dividing cells, i.e., cells with a number of midbody scars, arecancerous cells. For example, the percentage of dividing cells in thesample could be compared to a reference sample that represents thepercentage of dividing cells in a normal sample. Alternatively, otherindicia, such as morphology or the presence of cancer markers, can alsobe used to confirm a diagnosis of a proliferative disorder.

Midbody scars can also be used to isolate proliferating cells, e.g.,stem or progenitor cells, and grow them for a multitude of clinicalapplications including reversing, delaying the progression of, orpreventing degenerative diseases such as Alzheimer's and Parkinson'sdiseases, repairing damaged cardiac tissue after an ischemic injury,e.g., after a myocardial infarction, to provide dermal and epidermalprogenitor cells to regrow skin in subjects who are in need thereof,e.g., subjects with severe burns, and to target proliferating cells,e.g., cancer cells. Midbody scars can also be used for thecharacterization of such cells.

Alternatively, antibodies to components of the midbody scar can be usedto target toxins (e.g., proteins, radioactive molecules) to kill highlyproliferative cells, e.g., cancer or psoriasis cells, by linking toxinsto antibodies to extracellular components of the midbody scars, or a dyeor other label that can be bound to the cells that contain the scars andthen selectively heated/killed, e.g., by lasers tuned to the particularabsorption wavelength of the dye or label.

The methods described herein in which antibodies to midbody scarpolypeptides are employed may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one such specificantibody described herein, which may be conveniently used, for example,in clinical settings, to diagnose subjects exhibiting symptoms ofdisorders associated with aberrant expression of nucleic acids orpolypeptides.

In some aspects, a compound (e.g., an antibody or antigen-bindingfragment thereof) that binds to a midbody scar on a cell can be used inthe production of a composition, e.g., a therapeutic or diagnosticcomposition, by conjugating a therapeutic or diagnostic moiety such as adrug, toxin, chelator, a boron compound and a detectable label, to themidbody-scar binding compound. Methods for making such compositions areknown in the art, see, e.g., McCarron et al., Mol. Interv. 5(6):368-80(2005), and U.S. Pat. Pub. No. 2006/0088539. The compositions can beused to target drug molecules to cells that contain midbody scars. Insome embodiments, these compositions can be used to treat disordersassociated with cellular proliferation. Examples of cellularproliferative disorders include atherosclerosis, rheumatoid arthritis,idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver,lupus, vasculitis, endometriosis, uterine fibroids, prostatichyperplasia, nephritis, skin disorders, e.g., psoriasis, and cancer,e.g., carcinoma, sarcoma, metastatic disorders, or hematopoieticneoplastic disorders, e.g., leukemias. Specific cancers can includeprostate, breast, and ovarian cancers. In other embodiments, thesecompositions can be used to target diagnostic moieties to cells in vitroor in vivo that contain midbody scars.

For example, the drug moiety can be a protein or polypeptide possessinga desired biological activity. Such proteins include, for example,toxins such as abrin, ricin A, maytansinoids, Pseudomonas exotoxin, orDiphtheria toxin; proteins such as tumor necrosis factor,alpha-interferon, beta-interferon, nerve growth factor, platelet derivedgrowth factor, tissue plasminogen activator; and biological responsemodifiers such as lymphokines, interleukin-1, interleukin-2,interleukin-6, granulocyte macrophage colony stimulating factor,granulocyte colony stimulating factor, or other growth factors.Conjugated antibodies can be used to target drug molecules to cells thatcontain midbody scars. In one aspect, these antibodies can be used totreat disorders associated with cellular proliferation, e.g., cancers orpsoriasis.

Techniques for conjugating a therapeutic or diagnostic moiety to anantibody are well known (see, e.g., Arnon et al., 1985, “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al., Eds., Alan R.Liss, Inc. pp. 243-256; Hellstrom et al., 1987, “Antibodies For DrugDelivery”, in Controlled Drug Delivery, 2nd ed., Robinson et al., Eds.,Marcel Dekker, Inc., pp. 623-653; Thorpe, 1985, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., Eds., pp.475-506; “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al., Eds.,Academic Press, pp. 303-316, 1985; and Thorpe et al., 1982, Immunol.Rev., 62:119-158). Alternatively, an antibody can be conjugated to asecond antibody to form an antibody heteroconjugate as described bySegal in U.S. Pat. No. 4,676,980.

Pharmaceutical Formulations

Once a candidate compound that binds to a midbody scar has beenidentified, standard principles of medicinal chemistry can be used toproduce derivatives of the compound. Derivatives can be screened forimproved pharmacological properties, for example, efficacy,pharmaco-kinetics, stability, solubility, and clearance. The moietiesresponsible for a compound's activity in the assays described above canbe delineated by examination of structure-activity relationships (SAR)as is commonly practiced in the art. A person of ordinary skill inpharmaceutical chemistry could modify moieties on a candidate compoundor agent and measure the effects of the modification on the efficacy ofthe compound or agent to thereby produce derivatives with increasedpotency. For an example, see Nagarajan et al., J. Antibiot., 41:1430-8(1988). Furthermore, if the biochemical target of the compound (oragent) is known or determined, the structure of the target and thecompound can inform the design and optimization of derivatives.Molecular modeling software is commercially available (e.g., fromMolecular Simulations, Inc.) for this purpose.

The compounds and agents, nucleic acids, polypeptides, and antibodies(all of which can be referred to herein as “active compounds”), can beincorporated into pharmaceutical compositions. Such compositionstypically include the active compound and a pharmaceutically acceptablecarrier or excipient. A “pharmaceutically acceptable carrier” caninclude solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

There are a number of methods by which the new compositions for use inthe new methods can be delivered to subjects, in general, and tospecific cells or tissue in those subjects, in particular. In oneexample, plasmids encoding antibodies specific to midbody scarcomponents (e.g., intrabodies) can be injected into a tissue. Theplasmids would then enter cells in that tissue and express a specificantibody, which, in turn, would bind to the targeted midbody scarprotein. Delivery specificity of such plasmids can be enhanced byassociating them with organ- or tissue-specific affinity, so that theypreferentially enter specified cell types.

Compounds and their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral or rectaladministration.

The compounds will generally be formulated for parenteral administrationby injection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use. Where the compositions are intended for use in aspecific treatment area, e.g., for treating a tumor or psoriatic lesion,the compositions can be administered by one or more local injectionsinto the tumor site to diminish as much as possible any side effectsrelating to the compound's activities outside of the treatment area.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The therapeutic compositions of the invention can also contain a carrieror excipient, many of which are known to skilled artisans. Methods formaking such formulations are well known and can be found in, forexample, Remington: The Science and Practice of Pharmacy, University ofthe Sciences in Philadelphia (USIP), 2005.

The compositions can also be formulated for intracellular delivery ofthe active compounds, using methods known in the art. For example, thecompositions can include liposomes or other carriers that deliver theactive compound across the plasma membrane. Vesicles that are coveredwith membrane-permeant peptides, such as Tat or Antennapedia, can alsobe used. A number of other methods for enhancing intracellular deliveryare familiar to those of skill in the art.

It is recognized that the pharmaceutical compositions and methodsdescribed herein can be used independently or in combination with oneanother. That is, subjects can be administered one or more of thepharmaceutical compositions, e.g., pharmaceutical compositionscomprising a nucleic acid molecule or protein of the invention or amodulator thereof, subjected to one or more of the therapeutic methodsdescribed herein, or both, in temporally overlapping or non-overlappingregimens. When therapies overlap temporally, the therapies may generallyoccur in any order and can be simultaneous (e.g., administeredsimultaneously together in a composite composition or simultaneously butas separate compositions) or interspersed. By way of example, a subjectafflicted with a disorder described herein can be simultaneously orsequentially administered both a cytotoxic agent which selectively killsaberrant cells and an antibody (e.g., an antibody of the invention)which can, in one embodiment, be conjugated or linked with a therapeuticagent, a cytotoxic agent, an imaging agent, or the like.

EXAMPLES Example 1 An MKLP 1-Positive Ring Localizes at the MidbodyDuring Cytokinesis

During cytokinesis, a phase-dense material known as the midbody formsbetween the two daughter cells (FIG. 1A).

Diploid, telomerase-immortalized human RPE cells (hTERT-RPE-1s, ClontechLaboratories, Inc.; Morales et al., 1999, Nat. Genet., 21:115-118) andHeLa cells (American Type Culture Collection) were used in theexperiments described herein. All cells were grown using standardmethods, see, e.g., Gromley et al., 2003, J. Cell Biol., 161:535-545.HeLa cells were transfected as previously described (usingLipofectamine™ transfection reagent, Invitrogen). Cells undergoingcytokinesis were fixed and stained simultaneously with antibodies toMKLP1 and γ-tubulin, as well as 4′-6-Diamidino-2-phenylindole (DAPI),using standard methodology.

Antibodies to the following proteins were used in the Examples describedherein: α-tubulin and γ-tubulin (Sigma-Aldrich); Aurora B (TransductionLaboratories); MKLP-1 (Santa Cruz Biotechnology, Inc.); and GT335 forstabilized microtubules (Gromley et al., 2003, J. Cell Biol.,161:535-545).

Cells were prepared for immunofluorescence, imaged, and deconvolved(Metamorph, Universal Imaging Corp.) using either formaldehyde,formaldehyde followed by methanol, or methanol alone as previouslydescribed (Dictenberg et al., 1998, J. Cell Biol., 141:163-174). Allimmunofluorescence images are two-dimensional projections ofthree-dimensional images to ensure that all stained material was visiblein two-dimensional images. Quantification of signals produced byimmunofluorescence staining for various midbody antigens was performedas described for centrosome protein quantification (Gromley et al.,2003, J. Cell Biol., 161:535-545).

A ring of MKLP1 staining colocalized with the midbody structure, alongwith γ-tubulin (FIGS. 1B-1E). FIG. 1B depicts MKLP1 staining as a ringstructure associated with the midbody. FIG. 1C depicts ε-tubulinstaining, also as a ring structure associated with the midbody. FIG. 1Ddepicts DAPI staining of DNA. FIG. 1E is a merged combination of FIGS.1B-1D, in which MKLP1 staining is pseudo-colored in red, γ-tubulinstaining is pseudo-colored in green, and DAPI staining is pseudo-coloredin blue.

These results confirm the presence of MKLP1 that localizes at themidbody during cytokinesis, and demonstrate the presence of a ringstructure therein.

Example 2 A Midbody Scar Persists in Prophase Cells

A phase-dense midbody-like structure was observed in a prophase cell(FIG. 2A). This structure is likely to be the remnants of the midbodyring from a previous mitosis, referred to herein as a midbody scar. Aprophase cell was fixed and stained simultaneously with antibodies toMKLP1 and γ-tubulin, as well as DAPI. FIG. 2B depicts MKLP1 staining.FIG. 2C depicts γ-tubulin staining. FIG. 2D depicts DAPI staining ofDNA. FIG. 2E is a merged combination of FIGS. 2B-2D, in which MKLP1staining is pseudo-colored in red in the original, γ-tubulin staining ispseudo-colored in green in the original, and DAPI staining ispseudo-colored in blue in the original. The midbody scar is enlarged inthe insets. The midbody scar contained at least MKLP1 (FIGS. 2B and 2E)and Aurora B kinase, but lacked α-tubulin and γ-tubulin (FIGS. 2C and2E). This example demonstrates that a midbody-like structure containingMKLP1 and Aurora B kinase can be found in prophase cells.

Example 3 The Midbody Scar Persists During Mitosis

Cells in various stages of mitosis were fixed and stained to visualizeMKLP1, γ-tubulin, and DNA (FIGS. 3A ₁-3C₅). FIGS. 3A ₁-3A₅ depictmetaphase. FIGS. 3B ₁-3B₅ depict anaphase. FIGS. 3C ₁-3C₅ depicttelophase. FIGS. 3A ₁, 3B₁, and 3C₁ show phase contrast. FIGS. 3A ₂,3B₂, and 3C₂ depict γ-tubulin staining. FIGS. 3A ₃, 3B₃, and 3C₃ depictMKLP1 staining. FIGS. 3A ₄, 3B₄, and 3C₄ depict DAPI staining of DNA.FIGS. 3A ₅, 3B₅, and 3C₅ depict the merge of the three previous figures,with γ-tubulin pseudo-colored in green in the original, MKLP1pseudo-colored in red in the original, and DAPI pseudo-colored in bluein the original. Midbody scars were found in mitotic cells at all stagesof mitosis, including prometaphase, metaphase (FIGS. 3A ₃ and 3A₅),anaphase (FIGS. 3B ₃ and 3B₅), and telophase (FIGS. 3C ₃ and 3C₅).Additionally, the midbody scar was still observed when MKLP1 localizedto mitotic spindles. These findings indicate that the midbody scar is apersistent structure that is maintained through mitosis.

Example 4 Accumulation of Midbody Scars

Cells were also found to accumulate multiple midbody scars from multiplecell divisions. Interphase cells were fixed and stained to visualizeMKLP1, α-tubulin, and DNA. FIG. 4A depicts staining of α-tubulin. FIG.4B depicts staining of MKLP1. FIG. 4C depicts DAPI staining of DNA. FIG.4D is the merge of FIGS. 4A-4C, with α-tubulin pseudo-colored in greenin the original, MKLP1 pseudo-colored in red in the original, and DAPIpseudo-colored in blue in the original. FIGS. 4A-4D show an interphasecell that contains four visible midbody scars. This demonstrates thatafter multiple round of mitosis, midbody scars can be accumulated incells. At the end of cytokinesis, the midbody was found to retractpreferentially to one of the daughter cells.

Example 5 Segregation of Midbody Scars is Nonrandom in Tumor Cells andEmbryonic Stem Cells

During mitosis, if segregation of midbody scars was random, they wouldbe expected to be distributed equally between the two daughter cells.

Therefore, segregation of midbody scars was evaluated in human HeLacervical cancer cells and human H9 embryonic stem cells cultured bystandard methods, see, e.g., U.S. Pat. No. 7,005,252, using theimmunofluorescence and microscopy methods described herein.

Nonrandom (polarized) accumulation of midbody scars was observed in HeLatumor cells. In a sample of 145 cells in telophase, 40 (28%) showedequal distribution of the midbody scars to the daughter cells, while 105(72%) showed distribution to one cell. In telophase cells with 3 or 4scars, 67-72% show asymmetric inheritance (3,0 or 4,0) (n=1,234telophase cells counted). In another sample, 44% of the HeLa cells had1-3 rings/cell (n=4,678).

An even more marked non-random distribution pattern was observed in theH9 embryonic stem cells (shown in FIG. 5). In a population ofundifferentiated stem cells, 88% had 1-7 rings/cell (n=1,256), whereas9% of a population of cells allowed to differentiate in culture had 1ring/cell (n=987).

These results demonstrate that midbody scars distribute preferentiallyrather than randomly, and that dividing cells, e.g., stem cells,accumulate midbody scars at a much greater rate than differentiatedcells. Therefore, midbody scars are useful markers for dividing cells,e.g., stem or progenitor cells.

Example 6 Identification of Stem Cells in Tissue Culture

Cells are fixed with formaldehyde or methanol using standard protocolsand then stained with one or more antibodies to proteins listed inTable 1. A fluor-labeled primary antibody is used, or alternatively alabeled secondary antibody is added to visualize the primary. Standardimmunofluorescence methods are then used to image the midbody scars.

Example 7 Identification of Stem Cells in Tissue Sections

To identify stem cells in paraffin or frozen tissue sections, thesections are labeled with one or more antibodies as described herein.Then, a secondary antibody is used, e.g., a secondary antibody coupledto horse radish peroxidase (for paraffin sections) or coupled to afluorescent probe (for frozen sections). Then, the sections are washedand the presence of midbody scars detected using known methodsappropriate for the secondary antibody used.

Example 8 Isolating Midbody Scars

Known midbody isolation procedures can be adapted for use on mitoticcells to interphase cells to isolate midbody scars. These methods arepreferably used on cells with high numbers of midbody scars; these cellscan be selected as described herein, e.g., using a cell line stablyexpressing GFP-MKLP1, a midbody scar marker to label the scars. Cellswith high numbers of midbody scars can be isolated by flow cytometryusing known cytometry techniques. The methods can include depolymerizingthe actin (e.g., using cytochalasin B) and/or microtubule network (e.g.,nocodazole) to release the midbody scars from the plasma membrane.

MKLP1 is degraded in mitotic cells except for the midbodyscar-associated fraction, which persists in interphase cells. Midbodyscars are isolated by affinity chromatography, e.g., using antibodies toa polypeptide component of the midbody scars or to GFP, with or withoutadditional biochemical methods to enrich for midbody scars, such assucrose gradients, and gel filtration.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of detecting the presence of a midbody scar in a mammaliancell, the method comprising: contacting a mammalian cell suspected tohave a midbody scar with an antibody or antigen-binding portion thereofthat binds specifically to a polypeptide component of a midbody scarselected from the group consisting of; and detecting binding of theantibody to its antigen, wherein binding of the antibody indicates thepresence of a midbody scar in the mammalian cell.
 2. The method of claim1, wherein the polypeptide is selected from the group consisting ofmitotic kinesin-like protein 1 (MKLP1); Epsilon tubulin (ε-tubulin);Centrosomal Protein 55 kDa (Cep55); and Aurora B kinase.
 3. The methodof claim 1, comprising: contacting the mammalian cell with two or moreantibodies or antigen-binding portions thereof that each bindspecifically to a polypeptide component of a midbody scar, and detectingbinding of the two or more antibodies, wherein colocalized binding ofthe two or more of antibodies indicates the presence of a midbody scar.4. The method of claim 4, wherein the polypeptide is selected from thegroup consisting of mitotic kinesin-like protein 1 (MKLP1); Epsilontubulin (ε-tubulin); Centrosomal Protein 55 kDa (Cep55); and Aurora Bkinase.
 5. The method of claim 1, wherein the cell is in interphase. 6.The method of claim 1, wherein the mammalian cell is a fixed cell. 7.The method of claim 6, wherein the fixed cell is in a pathologyspecimen.
 8. The method of claim 1, wherein the cell is in tissue. 9.The method of claim 1, wherein the antibody or antigen-binding portionthereof is bound or linked to a detectable moiety.
 10. The method ofclaim 9, wherein detecting binding of the antibody to its antigencomprises detecting the detectable moiety.
 11. A method of identifying adividing cell in a population of mammalian cells, the method comprising:obtaining a population of mammalian cells comprising at least one cellsuspected of being a dividing cell; and detecting a cell having one ormore midbody scars, wherein a cell having one or more midbody scars isidentified as a dividing cell.
 12. The method of claim 11, wherein thepopulation of cells comprises dividing cells and non-dividing cells. 13.The method of claim 11, wherein the dividing cell is a cancerous cell.14. The method of claim 11, wherein the dividing cell is a stem orprogenitor cell.
 15. The method of claim 11, further comprisingidentifying cells having a desired number of midbody scars.
 16. Themethod of claim 15, wherein the desired number is 0 to 2 midbody scarsper cell.
 17. The method of claim 15, wherein the desired number is 4 ormore midbody scars per cell.
 18. The method of claim 15, furthercomprising selecting cells having the desired number of midbody scars.19. The method of claim 18, further comprising ablating the selectedcells.
 20. The method of claim 18, further comprising isolating theselected cells.
 21. The method of claim 11, wherein identifying a cellwith one or more midbody scars comprises optically detecting thepresence of a cellular structure having morphological characteristics ofa midbody scar.
 22. The method of claim 21, wherein optically detectingthe presence of a cellular structure comprises using phase contrastmicroscopy to analyze the cell.
 23. The method of claim 22, wherein theanalysis is automated.
 24. The method of claim 11, wherein identifying acell with one or more midbody scars comprises contacting the populationof cells with a detectible antibody or antigen-binding portion thereofthat binds specifically to a polypeptide component of a midbody scar;and detecting binding of the detectible antibody to the polypeptide,wherein binding of the antibody indicates the presence of a midbodyscar.
 25. The method of claim 24, wherein the polypeptide component of amidbody scar is selected from the group consisting of mitotickinesin-like protein 1 (MKLP1); Epsilon tubulin (ε-tubulin); CentrosomalProtein 55 kDa (Cep55); and Aurora B kinase.
 26. The method of claim 24,wherein the detectible antibody or antigen binding portion thereofcomprises a fluorescent label.
 27. The method of claim 26, whereinidentifying a cell with one or more midbody scars comprises detectingthe presence of fluorescence from the detectible antibody or antigenbinding portion thereof.
 28. The method of claim 27, wherein detectingthe presence of fluorescence comprises using fluorescence microscopy.29. The method of claim 24, wherein the detection is automated.
 30. Themethod of claim 24, wherein the detectible antibody comprises an enzyme,which when provided with an appropriate substrate produces a productthat is detected.
 31. The method of claim 11, wherein the population ofcells is from a subject suspected of having a proliferative disorder.32. The method of claim 31, further comprising: determining thepercentage of cells in the sample that have a selected number of midbodyscars; and comparing the percentage of cells in the sample to areference percentage representing a sample from a subject that does nothave a proliferative disorder; wherein the presence of a significantlyhigher number of dividing cells as compared to the reference sampleindicates that the subject has a proliferative disorder.
 33. The methodof claim 31, wherein the proliferative disorder is selected from thegroup consisting of atherosclerosis, rheumatoid arthritis, idiopathicpulmonary fibrosis, scleroderma, cirrhosis of the liver, lupus,vasculitis, endometriosis, uterine fibroids, prostatic hyperplasia,nephritis, skin disorders, and cancer.
 34. The method of claim 11,further comprising isolating the dividing cells.
 35. The method of claim34, further comprising expanding the isolated dividing cells in cultureunder conditions sufficient to provide a population of dividing cells.36. An isolated midbody scar from a mammalian cell, wherein the midbodyscar comprises mitotic kinesin-like protein 1 (MKLP1); Epsilon tubulin(ε-tubulin); Centrosomal Protein 55 kDa (Cep55); and Aurora B kinase.